Lesson 15 Flashcards
in sickle cell disease, what type of mutation causes disease?
a single point mutation
what specific point mutation causes sickle cell disease?
the substitution of glutamic acid with a valine in position 6, giving rise to a mutant global chain called HbS (sickle)
what does HbS cause in erythrocytes?
in deoxygenation where HbS forms fibers (different from normal Hb which is present as globular) in a process called polymerization → these fibers are toxic inside erythrocytes thus damaging them, causing them to lose cations and water forming the sickle shape
what are the major symptoms of sickle cell disease?
hemolytic anemia, vaso-occlusion, and morbidity and mortality
what is vaso occlusion?
impaired rheology, adhesion between sickled erythrocytes, neutrophils, endothelium, and platelets
what is hemolysis?
results in the release of the heme group, which is toxic and induces endothelial dysfunction (particularly toxic in the small vessels because damages the vessel walls, the endothelium), the release of cytokines, which results in an inflamed microenvironment, a sterile inflammation
what other disease does the geographic distribution of sickle cell disease correlate with?
malaria
what is the effect of hemolytic anemia?
blocks the small blood vessels
what does vaso occlusion cause systemically?
causes ischemic damage to different tissues including the nervous system leading to stroke
what are the chronic complications of sickle cell disease?
general vasculopathy and progressive ischemic organ damage in important organs like the kidneys, bones and liver
currently what are the only two treatments for sickle cell disease?
bone marrow transplant or blood transfusion
what does HPFH stand for?
hereditary persistence of fetal hemoglobin
what is HPFH?
associated with high levels of fetal hemoglobin in adults (normally in adults we undergo a globin switch so we do not have any fetal hemoglobin)
in what case is HPFH benign?
when the 𝛾-globin gene is reactivated
what are the two molecular causes of HPFH?
point mutations in the HGB gene or locus (promotor of the 𝛾-globin) or deletions in the HGB gene or locus downstream of the 𝛾-globin gene
what do point mutations in the HGB gene lead to?
non-deletional HPFH → in the γ-promoter we have binding sites for repressors: some mutations in here cause disruption of the repressor binding site ; there could also be the creation of an activator binding site
what do deletions in the HGB gene cause?
lead to deletional HPFH → these kinds of mutations are not really fully explained - however, in some cases there is a deletion of repressor binding sites, in some other cases it’s not clear why the deletion causes HPFH
what can we induce for the reactivation of the 𝛾-globin gene?
induce NHEJ (non-homologous end joining) and disrupt the downstream gene, which is way easier → you can delete the gene for the repressor if you don’t want to have repression
what three regions were associated to having high levels of HbF in HPHF patients?
- In an intergenic region between HBS1L and MYB on chromosome 6
- Inside the β-locus, some regions are responsible for reactivating γ-globin
- A new candidate, which is the BCL11A gene, located on chromosome 2
what form of BCL11A has the ability to silence 𝛾 genes?
the erythroid form
which other repressors form a complex with BCL11A to silence the 𝛾-genes?
LRF, GATA1, FOG1, and NuRD
in what four ways can we induce HPFH?
- target the BCL11A erythroid enhancer
- induction of non-deletional HPFH
- induction of deletional HPFH
- gene repair
how can we target the BCL11A erythroid enhancer?
we can delete the enhancer with zinc finger or Cas to obtain less BCL11A expression leading to the reactivation and expression of 𝛾 genes
how can we induce non-deletional HPFH?
by creating mutations on the binding sites on the promotors of 𝛾-genes to not allow the binding of repressors
how can we induce deletional HPFH?
using zinc finger, Cas9, or NHEJ to delete the repressors binding sites resulting in big deletions
how can we use gene repair to induce HPFH?
we can induce the repair of the mutated gene through HDR (clinically speaking currently this is not really possible since HDR occurs at very low frequencies)
how can HbF be therapeutic?
it has anti-sickling properties as HbF is able to repel HbS polymerization → the presence of HbF impedes the polymerization of HbS
what is an important characteristic of HbF?
it is an anti-sickling molecule
how can we use gene correction to treat β-thalassemia?
with gene correction we have low efficiency in hematopoietic stem cells (HSCs), meaning that a lot of work is still ongoing, because this will be a
very valid approach to replace the mutated genes, so not the aim is to optimize the reagents to achieve higher efficiency
how was targeting used to treat β-thalassemia with gene correction?
a specific place in the Hb locus was targeted (more than just a replacement)
how was the Hb locus targetted, and what was this the first use of?
the first use of ribonuclear particle, instead of just mRNA, so in this case it was clear that the efficiency of indels (of
cutting) was much higher if I provided all the system using Rnp instead of just mRNA.
describe enhancer disruption in BCL11A when treating β-thalassemia:
in this case we have a completely different realist because the efficiency of non-homologous end joining is very high
how does enhancer disruption work?
- one is disrupting a the coding part of the gene, and the gene editing tool used in this case are the ZFNs homologous to the coding region
- another couple of ZFNs targets the enhancer sequence (so not the coding sequence, just in a regulatory element promoting the expression of BCL11A in the erythroid lineages)
what is an important step to consider when working with ZFN or with any other gene editing strategies?
you must be aware of off targets → the
system has to be very specific in order to cut the human genome just in the desired region
how do ZFNs effect the genome in enhancer disruption?
most of the genome editing sequence give a KO because most of them are deletion that make the sequence go out of frame
if you eliminate the sequence. what occurs?
you will not have expression of BCL11A in al the cells in which it is expressed, while targeting the specific erythroid enhancer we delete it only from erythroid cells
how is genome editing used to reproduce and generate the naturally occurring mutations associated with HPFH?
mutation in the γ promoter creates an E-box motif which composes a consensus sequence for an activator,
which is the TAL1 erythroid specific activator, created on the antisense strand of the γ-globin promoter
how is genome editing used to reproduce and generate the naturally occurring mutations associated with HPFH?
mutation in the γ promoter creates an E-box motif which composes a consensus sequence for an activator,
which is the TAL1 erythroid specific activator, created on the antisense strand of the γ-globin promoter
how is genome editing used to reproduce and generate the naturally occurring mutations associated with HPFH?
mutation in the γ promoter creates an E-box motif which composes a consensus sequence for an activator,
which is the TAL1 erythroid specific activator, created on the antisense strand of the γ-globin promoter
what does the deletional HPFH mutation copy?
the 13 nucleotide deletion → done in HUDEP cell line
out of all all the ways to induce HPFH, what is the most effective?
the elimination of the erythroid specific BCL11A expression
what was the specific goal of both clinical trials to treat β-thalassemia and sickle cell disease?
to elevate HbF to achieve a therapeutic effect
what occurred in the CTX001 clinical trial?
received autologous CD34+ cells but
cells were modified with Crispr-Cas9 and the guide targeting erythroid specific lineage enhancer BLC11A
(instead of lentiviral vectors)
what was the CTX001 clinical trial the first to use?
genome editing with CRISPR-Cas9 ex vivo via transplantation into the patient
what is the rational in the CTX001 study?
disrupt GATA1 binding site in the BCL11A promotor only present in the erythroid lineage
what is different about gene editing in animal models compared to most other experiments?
genome editing works well in human cells but not so good in murine cells - one of the few cases where a technique works better in humans than in animal clinical trials
for the β-thalassemia patients, what was the outcome of the clinical trial?
no more transfusions were needed in the 20 months of follow up → the level of adult Hb decreases, and HbF starts to appear and increases in quantity from the 2nd month to the 18th, while the level of transfused Hb keeps fading away because red cells die in the meantime
what was the result of the CTZ001 clinical trial in SCD patients?
we can notice a big quantity of HbA because the patients are transfused a lot after the transplantation; after that, levels of HbF starts to grow up, even though we’re not eliminating sickling Hb, but just diluting it: the dilution has anti-sickling activity. That was enough in this patient, that indeed did not
experience any vaso-occlusive crisis in the follow up
